CN115792007A - Method for determining content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography - Google Patents
Method for determining content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography Download PDFInfo
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Abstract
The invention relates to a method for measuring the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography. Precisely adding 9mL of sample to be tested and 1mL of acid reagent, heating at 80 ℃ for 30min in a graphite digestion instrument, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring with purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, standing for layering, taking water phase, filtering, and taking filtrate to obtain sample solution to be tested. Respectively and precisely measuring 0.1mL of each of the aluminum element series reference solution, the blank reference solution, the sample solution to be tested and the blank sample solution, respectively and precisely adding 0.9mL of the derivative reagent, respectively, uniformly mixing, respectively injecting into a liquid chromatograph, and recording a chromatogram. According to the method, a proper demulsifying acid solvent is selected to destroy the oil-in-water structure of the fat emulsion, so that the wrapped impurity aluminum element is released, and a clear solution is obtained at the same time, so that the requirement of high performance liquid chromatography sample injection is met.
Description
Technical Field
The invention belongs to the field of medicine detection, and particularly relates to a method for determining the content of impurity aluminum element in fat emulsion injection based on high performance liquid chromatography.
Background
Aluminum is one of the most abundant metal elements in the earth's crust. The accumulation of aluminum in the human body increases the risk of disease. The determination of impurity aluminum element in large-volume injection has received extensive attention. In the prior art, methods for detecting aluminum in medicines mainly comprise the following steps: the fat emulsion injection comprises a colorimetric method, an atomic absorption spectrum, an inductively coupled plasma atomic emission spectrometry (ICP-AES), an inductively coupled plasma mass spectrometry (ICP-MS) and the like, wherein the fat emulsion injection has more complex components, more diversified forms of aluminum elements and low sensitivity of the colorimetric method; the fat emulsion is easy to carbonize at high temperature, and an atomic absorption spectrometry method is not suitable; ICP-AES and ICP-MS instruments are expensive, and are not favorable for popularization and use in laboratories. The fat emulsion injection is a large-capacity parenteral nutrition injection widely applied to clinic, and the possible impurity aluminum element can directly enter blood and accumulate in a human body to cause more harm to the human body, especially to renal insufficiency patients and infants.
The high performance liquid chromatography has the advantages of high speed, high pressure, high efficiency, high sensitivity, wide application range, reusable column, small sample amount and the like. However, the fat emulsion injection has the following difficulties that the impurity aluminum element is still blank in the detection of the high performance liquid chromatography: (1) The fat emulsion injection has complex composition, can not be directly injected, can not be simply diluted by purified water or solvent for injection, and can cause the blockage of a chromatographic column and a high-performance liquid phase system. (2) The content of aluminum element impurities in the sample is low (the limit of the aluminum element in parenteral nutrition injection specified by United states pharmacopoeia is not more than 25 mu g/L), so the sample introduction requirement cannot be met by increasing the dilution times, otherwise the detection limit and the quantification limit of the determination method cannot meet the determination requirement. (3) The oil-in-water structure of the fat emulsion is likely to wrap aluminum element which is derived from raw materials, so that the measurement result is inaccurate.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for determining the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography, which comprises the steps of selecting a proper solvent to destroy the structure of fat emulsion oil-in-water on the premise of meeting the requirements of quantitative limit and detection limit of the high performance liquid chromatography, releasing the wrapped impurity aluminum element, obtaining a clear solution and meeting the requirement of high performance liquid chromatography sample injection.
The technical scheme adopted by the invention is as follows: the method for determining the content of the impurity aluminum element in the fat emulsion injection by the high performance liquid chromatography comprises the following steps:
1) Preparing a reagent: preparing a derivative reagent, an aluminum element series reference substance solution, a blank reference solution and a mobile phase respectively, and preparing the solution on site;
2) Preparing a sample solution of a test sample: taking a polytetrafluoroethylene digestion tube, precisely adding 9mL of a sample to be tested and 1mL of an acid reagent, plugging, placing the tube into a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring with purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, shaking up, standing for layering, removing an oil phase, filtering a water phase, and taking a filtrate to obtain a sample solution of the sample to be tested; the sample to be tested is fat emulsion injection;
3) Preparing a blank test solution: taking a polytetrafluoroethylene material digestion tube, precisely adding 9mL of purified water and 1mL of acid reagent, plugging, placing in a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring with purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, shaking up, standing for layering, taking out the lower-layer water phase for filtration, taking the filtrate, and obtaining a blank sample solution;
4) And (3) determination: respectively and precisely measuring 0.1mL of each of an aluminum element series reference solution, a blank reference solution, a sample solution to be tested and a blank sample solution, respectively and precisely adding 0.9mL of a derivatization reagent, respectively, uniformly mixing, respectively injecting into a liquid chromatograph, and recording a chromatogram;
5) And (3) performing linear regression by subtracting the peak area of the blank control solution from the corresponding peak area of the concentration of the aluminum element series control solution according to the concentration of the aluminum element series control solution to obtain a linear regression equation, and subtracting the peak area of the blank test solution from the peak area of the test sample solution to calculate the content of the aluminum element in the test sample.
Preferably, in step 1), the method for preparing the derivatizing reagent comprises the following steps: according to the volume ratio, the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.3 percent, and the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.2mol/L ammonium acetate solution = 45; taking 50mL of a mixed solution of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2mol/L ammonium acetate solution, adding 0.3mL of NaOH with the mass percentage concentration of 50%, and uniformly mixing.
Preferably, in the step 1), the concentrations of the aluminum element series reference substance solution are respectively 200 mug/L, 100 mug/L, 50 mug/L, 25 mug/L, 10 mug/L and 5 mug/L.
Preferably, in step 1), the method for preparing the mobile phase comprises the following steps: according to the volume ratio, the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.3 percent, and the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.2mol/L ammonium acetate solution = 45; taking 0.3 percent of 8-hydroxyquinoline acetonitrile solution and 0.2mol/L of ammonium acetate solution, and uniformly mixing.
Preferably, in step 2), the acid reagent is sulfuric acid, nitric acid or hydrochloric acid.
More preferably, the acid agent is hydrochloric acid.
Preferably, in step 3), the liquid chromatography conditions are: adopting a phenyl chromatographic column with phenethyl bonded silica gel as a matrix, wherein the column temperature is 30 ℃; the flow rate is 1.0mL/min; the sample injection volume is 100 mu L; the excitation wavelength of the fluorescence detector was 380nm and the emission wavelength was 520nm.
Preferably, the fat emulsion injection is C 6 -C 24 The medium-long chain fat emulsion injection, or the fat emulsion injection containing fat emulsion amino acid (17) and glucose (11%), or the medium-long chain fat emulsion injection containing fat emulsion amino acid (16) and glucose (16%).
The method provided by the invention is applied to determination of the content of impurity aluminum element in fat emulsion injection.
The invention has the beneficial effects that:
1. according to the method, a proper demulsifying acid solvent is selected to destroy the oil-in-water structure of the fat emulsion, so that the wrapped impurity aluminum element is released, and a clear solution is obtained at the same time, so that the requirement of high performance liquid chromatography sample injection is met.
2. The method provided by the invention optimizes the pretreatment conditions of the sample, reduces the introduction of aluminum element, and improves the accuracy of the determination of impurity aluminum element in the sample.
3. The method of the invention selects proper pretreatment method and chromatographic conditions, and realizes the determination of the content of impurity aluminum element in the fat emulsion injection by high performance liquid chromatography.
Drawings
FIG. 1 is a high performance liquid chromatogram of example 1, in which the acid solvent is sulfuric acid and the volume ratio of the sample to the sulfuric acid is 9.
FIG. 2 is a high performance liquid chromatogram of example 1, in which the acid solvent is sulfuric acid and the volume ratio of the sample to the sulfuric acid is 8.
FIG. 3 is a high performance liquid chromatogram of example 1, in which the acid solvent is hydrochloric acid and the volume ratio of the sample to the hydrochloric acid is 9.
FIG. 4 is a high performance liquid chromatogram of example 1, in which the acid solvent is hydrochloric acid and the volume ratio of the sample to the hydrochloric acid is 8.
FIG. 5 is a high performance liquid chromatogram of example 1, wherein the acid solvent is nitric acid, and the volume ratio of the sample to the nitric acid is 9.
FIG. 6 is a high performance liquid chromatogram of example 1, in which the acid solvent is nitric acid and the volume ratio of the sample to the nitric acid is 8.
FIG. 7 is a high performance liquid chromatogram of example 1 in which the alkali solvent was NaOH.
FIG. 8 is a high performance liquid chromatogram of example 1, in which the acid solvent is hydrochloric acid and the temperature is 60 ℃ in a water bath for 5 hours.
FIG. 9 is a high performance liquid chromatogram of example 1, in which the acid solvent is hydrochloric acid and the temperature is 70 ℃ in a water bath for 5 hours.
FIG. 10 is a high performance liquid chromatogram obtained in example 1, in which the acid solvent is hydrochloric acid and the temperature is 80 ℃ in a water bath for 5 hours.
FIG. 11 is a high performance liquid chromatogram obtained by n-heptane extraction in example 1, in which the acid solvent was hydrochloric acid, the graphite digestion apparatus was heated.
FIG. 12 is a standard curve of the control solution of the aluminum element series in example 3.
Detailed Description
The method for determining the content of the impurity aluminum element in the fat emulsion injection by the high performance liquid chromatography comprises the following steps:
1) Preparing a reagent:
and respectively preparing a derivative reagent, an aluminum element series reference substance solution, a blank reference solution and a mobile phase, and preparing the solution on site.
1.1 Formulation of derivatizing reagents: according to the volume ratio, the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.3 percent, and the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.2mol/L ammonium acetate solution = 45; taking 50mL of a mixed solution of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2mol/L ammonium acetate solution, adding 0.3mL of NaOH with the mass percentage concentration of 50%, and uniformly mixing.
1.2 Preparation of aluminum element series control solutions: the concentrations of the aluminum element series reference substance solutions are respectively 200 mug/L, 100 mug/L, 50 mug/L, 25 mug/L, 10 mug/L and 5 mug/L.
1.3 Blank control solution): purified water was used as a blank solution.
1.4 Preparation of mobile phase: according to the volume ratio, the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.3 percent, and the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.2mol/L ammonium acetate solution = 45; 0.3 percent of 8-hydroxyquinoline acetonitrile solution and 0.2mol/L of ammonium acetate solution are mixed evenly.
2) Preparing a sample solution of a test sample:
taking a polytetrafluoroethylene material digestion tube, precisely adding 9mL of a sample to be tested and 1mL of an acid reagent, plugging, placing the tube into a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring by using purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, shaking uniformly, standing for layering, removing an oil phase, filtering a water phase, and taking a filtrate to obtain a sample solution of the sample to be tested; the sample to be tested is fat emulsion injection.
The acid reagent is sulfuric acid, nitric acid or hydrochloric acid. Preferably, the acid agent is hydrochloric acid.
3) Blank test solution:
taking a polytetrafluoroethylene digestion tube, precisely adding 9mL of purified water and 1mL of acid reagent, plugging, placing in a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring with purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, shaking up, standing for layering, taking a lower-layer water phase, filtering, taking a filtrate, and obtaining a blank sample solution.
The acid reagent is sulfuric acid, nitric acid or hydrochloric acid. Preferably, the acid agent is hydrochloric acid.
4) And (3) determination:
respectively and precisely measuring 0.1mL of each of the aluminum element series reference solution, the blank reference solution, the sample solution to be tested and the blank sample solution, respectively and precisely adding 0.9mL of the derivative reagent, respectively, uniformly mixing, respectively injecting into a liquid chromatograph, and recording the chromatogram.
The liquid chromatography conditions were: adopting a phenyl chromatographic column with phenethyl bonded silica gel as a matrix, wherein the column temperature is 30 ℃; the flow rate is 1.0mL/min; the sample injection volume is 100 mu L; the excitation wavelength of the fluorescence detector was 380nm and the emission wavelength was 520nm.
5) Drawing a standard curve and calculating:
and (3) performing linear regression by subtracting the peak area of the blank control solution from the corresponding peak area of the concentration of the aluminum element series control solution to obtain a linear regression equation, and calculating the content of the aluminum element in the sample to be tested according to the peak area of the sample solution to be tested by subtracting the peak area of the blank sample solution.
Example 1
The present example uses the following control and test samples for the conditional screening test.
Comparison products: the concentration of standard solution of metallic aluminum single element, national analysis and test center of nonferrous metals and electronic materials is 1000 mug/mL.
Sample of the test article: medium-long chain fat emulsion injection (C) 6 -C 24 ) Liaoning Haishiko pharmaceuticals, inc.
Selection of solvent for destroying oil-in-water structure of fat emulsion
1. Organic solvent
As shown in Table 1, ethanol, isopropanol, n-propanol, tetrahydrofuran, n-hexane or a mixture of two reagents in a certain volume ratio is respectively selected as an organic solvent to be used as a sample of a test sample to carry out a demulsification test, and the condition of destroying an oil-in-water structure is researched, and the results are shown in Table 1.
TABLE 1 demulsification Condition screening (organic solvent)
As can be seen from Table 1, ethanol, isopropanol, n-propanol, n-hexane, etc. did not destroy the oil-in-water structure. Tetrahydrofuran can destroy the oil-in-water structure, but has a large dilution factor. However, the aluminum content in the sample to be tested is usually low, and the sample is pretreated by a large dilution factor, which may cause the final sample injection concentration to be low, and the requirements of the detection limit and the quantification limit of the method cannot be met. Therefore, the organic solvent cannot be used as a demulsifying solvent for the fat emulsion injection of the present invention.
2. Acid reagent
Sulfuric acid, hydrochloric acid and nitric acid were respectively selected as acid reagents.
Derivatization reagent: 0.3% by volume of a solution of 8-hydroxyquinoline in acetonitrile: 0.2mol/L of ammonium acetate = 45; and (3) taking 50mL of a mixed solution of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2mol/L ammonium acetate solution, adding 0.3mL of NaOH with the mass percentage concentration of 50%, and uniformly mixing.
Preparing a sample solution of a test sample: taking a polytetrafluoroethylene material digestion tube, precisely adding 8mL of sample to be tested and 2mL of acid reagent or precisely adding 9mL of sample to be tested and 1mL of acid reagent, heating in a water bath at 60 ℃ for 3h to respectively obtain sample solutions of the sample to be tested with different volume ratios of the acid reagents, and observing the demulsification condition.
Precisely measuring the sample solutions of the test sample with different acid reagents and different volume ratios to be 0.1mL respectively, precisely adding 0.9mL of derivative reagent respectively, mixing uniformly, injecting into a liquid chromatograph respectively, and recording a chromatogram. The liquid chromatography conditions were: adopting a phenyl chromatographic column with phenethyl bonded silica gel as a matrix, wherein a mobile phase is a mixture of an 8-hydroxyquinoline acetonitrile solution with the mass percentage concentration of 0.3% and an ammonium acetate solution with the concentration of 0.2mol/L according to the volume ratio of 45; the column temperature is 30 ℃; the flow rate is 1.0mL/min; the sample injection volume is 100 mu L; the excitation wavelength of the fluorescence detector was 380nm and the emission wavelength was 520nm. The peak shape and the corresponding condition were analyzed by HPLC analysis, and the results are shown in Table 2 and FIGS. 1 to 6.
3. Alkali reagent
NaOH is used as an alkali reagent.
Derivatizing reagent: 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2mol/L ammonium acetate solution = 45; and (3) taking 50mL of a mixed solution of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2mol/L ammonium acetate solution, adding 0.3mL of NaOH with the mass percentage concentration of 50%, and uniformly mixing.
Preparing a sample solution of a test sample: taking a polytetrafluoroethylene digestion tube, precisely adding 5mL of a sample to be tested and 0.5mg of sodium hydroxide, heating in a water bath at 60 ℃ for 3h to obtain a sample solution of the sample to be tested, and observing the demulsification condition.
Precisely measuring 0.1mL of sample solution of the test sample, precisely adding 0.9mL of derivative reagent, mixing, injecting into a liquid chromatograph, and recording the chromatogram. The liquid chromatography conditions were: adopting a phenyl chromatographic column with phenethyl bonded silica gel as a matrix, wherein a mobile phase is a mixture of an 8-hydroxyquinoline acetonitrile solution with the mass percentage concentration of 0.3% and an ammonium acetate solution with the concentration of 0.2mol/L according to the volume ratio of 45; the column temperature is 30 ℃; the flow rate is 1.0mL/min; the sample injection volume is 100 mu L; the excitation wavelength of the fluorescence detector was 380nm and the emission wavelength was 520nm. The peak shape and the corresponding condition were analyzed by HPLC analysis, and the results are shown in Table 2 and FIG. 7.
TABLE 2 demulsification Condition screening (acid and base reagents combined HPLC analysis)
As can be seen from table 2, fig. 4 and fig. 6, when the volume ratio of the sample to be tested to the acid reagent is 8. After filtration by a 0.45 mu m filter membrane and sample injection by high performance liquid chromatography, the result shows that the response of the sample is lower after the sulfuric acid demulsification; the peak front edge of the sample after demulsification by hydrochloric acid and nitric acid has poor peak type. The peak pattern was not improved by adjusting the amount of base added to the derivatizing agent.
As can be seen from Table 2, FIG. 1, FIG. 3 and FIG. 5, when the volume ratio of the sample to be tested to the acid reagent is 9, the sample can be demulsified to reach the state of oil-water separation, and after filtration through a 0.45 μm filter membrane and sample injection by high performance liquid chromatography, the results show that the sample response is low after sulfuric acid demulsification, the sample peak is bifurcated after nitric acid demulsification, the peak profile is poor, and the sample peak profile is good after hydrochloric acid demulsification.
As can be seen from Table 2 and FIG. 7, the samples of the samples to be tested are demulsified after being added with sodium hydroxide to reach the state of oil-water separation, and the response value is too high through high performance liquid chromatography sample injection analysis, because the sodium hydroxide reagent contains a certain amount of aluminum elements, and foreign aluminum element impurities can be introduced by adding the sodium hydroxide.
In conclusion, the invention preferably selects hydrochloric acid as the acidification demulsification reagent, and the volume ratio of the sample to be tested to the hydrochloric acid is 9.
(II) screening of sample pretreatment heating conditions
The pretreatment process of the sample is screened by measuring the treated sample of the sample by an HPLC method, and analyzing the conditions of the sample, the standard sample and a standard curve by combining the sample.
1. Effect of Water bath heating on assay results
1.1 Preparation of reagents
Aluminum element standard stock solution: taking the standard solution of the metallic aluminum single element, and diluting the standard solution with purified water to prepare the standard stock solution of the aluminum element with the concentration of 500 mu g/L.
Preparing aluminum element series reference substance solution: precisely measuring a proper amount of standard stock solution of the aluminum element, adding purified water for quantitative dilution, and respectively preparing aluminum element series reference substance solutions with the concentrations of 200 mu g/L, 100 mu g/L, 50 mu g/L, 25 mu g/L, 10 mu g/L and 5 mu g/L.
Blank control solution: purified water was used as a blank solution.
Sample solution of test article: taking a 15mL polypropylene graduated centrifuge tube, adding 4.5mL of a sample to be tested and 0.5mL of hydrochloric acid according to the volume ratio of 9 to 1, sealing, placing in a water bath, heating at 60 ℃,70 ℃ and 80 ℃ for 3-5h respectively, taking out, and filtering to obtain sample solutions of the sample to be tested obtained at different temperatures.
Adding a standard sample solution: precisely measuring 4.5mL of standard stock solution of aluminum element with the concentration of 500 mug/L, adding 85.5mL of sample solution of a sample to be tested, and uniformly mixing to obtain a standard sample. Taking a 15mL polypropylene graduated centrifuge tube, adding 4.5mL of the standard sample and 0.5mL of hydrochloric acid according to a volume ratio of 9.
Derivatizing reagent: 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2mol/L ammonium acetate solution = 45; and (3) taking 50mL of a mixed solution of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2mol/L ammonium acetate solution, adding 0.3mL of NaOH with the mass percentage concentration of 50%, and uniformly mixing.
1.2 Method of)
Respectively and precisely measuring 0.1mL of an aluminum element series reference substance solution, a blank reference solution, a sample solution to be tested obtained at different temperatures and a standard sample solution obtained at different temperatures, respectively and precisely adding 0.9mL of a derivative reagent, uniformly mixing, respectively injecting into a liquid chromatograph, and recording a chromatogram.
The liquid chromatography conditions were: adopting a phenyl chromatographic column with phenethyl bonded silica gel as a matrix, wherein a mobile phase is a mixture of an 8-hydroxyquinoline acetonitrile solution with the mass percentage concentration of 0.3% and an ammonium acetate solution with the concentration of 0.2mol/L according to the volume ratio of 45; the column temperature is 30 ℃; the flow rate is 1.0mL/min; the sample injection volume is 100 mu L; the excitation wavelength of the fluorescence detector was 380nm and the emission wavelength was 520nm.
And (3) performing linear regression by using the corresponding peak areas of the concentrations of the aluminum element series reference substance solutions to obtain a linear regression equation, and respectively calculating the content of the aluminum element in the solutions according to the peak areas of the sample solutions of the samples to be tested obtained at different temperatures and the standard samples at different temperatures. The recovery was calculated and the results are shown in table 3.
Table 3 water bath heating condition screening:
as can be seen from figures 8-10, hydrochloric acid is selected as the demulsifying solvent for destroying the oil-in-water structure, and the chromatographic peak shape is better. However, as can be seen from table 3, the recovery was too high and the deviation between the parallel samples was large. This is because the hydrochloric acid is added in a small amount, and the oil-in-water structure is destroyed by heating, but the water phase and the oil phase are not completely miscible, which results in inaccurate measurement results and a risk of clogging in the liquid phase system. Meanwhile, the water bath heating temperature is low, and the time for destroying the oil-in-water structure is long. The temperature is high, the heating time is long, the liquid amount has certain loss, and the steam environment of the water bath has certain influence on the sample.
Combining the above factors, the water bath heating results in high recovery rate of the overall result, which is not suitable for the present invention.
2. Heating graphite digestion instrument, selection of organic solvent extractant
In order to shorten the extraction time and increase the efficiency of the solvent for destroying the emulsion structure, and simultaneously, considering the factors of completely dissolving the aluminum element in the water phase and the like, a graphite digestion instrument in an anhydrous environment is adopted as a heating device. Because the impurity aluminum element exists in the aqueous solution in the form of aluminum ions, an organic solvent extraction step is added, so that oil phase substances are completely dissolved in the extraction solvent and are completely separated from the aqueous phase, thereby avoiding the oil phase substances from entering the liquid phase system to influence the result accuracy and increasing the risk of blocking the liquid phase system.
2.1 Preparation of reagents
Standard stock solution of aluminum element: taking the standard solution of the metallic aluminum single element, and diluting the standard solution with purified water to prepare the standard stock solution of the aluminum element with the concentration of 500 mu g/L.
Preparing aluminum element series reference substance solution: precisely measuring a proper amount of standard stock solution of the aluminum element, adding purified water for quantitative dilution, and respectively preparing aluminum element series reference substance solutions with the concentrations of 200 mu g/L, 100 mu g/L, 50 mu g/L, 25 mu g/L, 10 mu g/L and 5 mu g/L.
Blank control solution: purified water was used as a blank solution.
Sample solution of test sample: taking a polytetrafluoroethylene digestion tube, adding 9mL of a sample to be tested and 1mL of hydrochloric acid according to the volume ratio of 9 to 1, plugging, placing in a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, destroying the oil-in-water structure of the fat emulsion, enabling the solution to be in a turbid state of oil-water separation, concentrating at 120 ℃, cooling, and transferring purified water to a constant volume of 10mL. Adding the extracting agents respectively as shown in Table 4, shaking up, standing for layering, discarding oil phase, and filtering water phase to obtain sample solutions of the test samples obtained by different extracting agents.
Adding a standard sample solution: 4.5mL of standard aluminum element stock solution with the concentration of 500 mu g/L is precisely measured, 85.5mL of sample solution of a sample to be tested is added, and the standard sample is uniformly mixed to obtain a standard sample. Taking a polytetrafluoroethylene digestion tube, adding 9mL of the standard sample and 1mL of hydrochloric acid according to a volume ratio of 9 to 1, adding a plug, placing the tube in a graphite digestion instrument, heating the tube at 80 ℃ for 30min, heating the tube to 100 ℃ for 1h, destroying the oil-in-water structure of the fat emulsion, concentrating the solution at 120 ℃ in an oil-water separation turbid state, cooling the solution, transferring the solution by using purified water, and fixing the volume to 10mL. As shown in Table 4, the extractant was added, shaken, and the aqueous phase was filtered to prepare standard samples containing 25. Mu.g/L of standard aluminum element.
Derivatizing reagent: 0.3% by volume of a solution of 8-hydroxyquinoline in acetonitrile: 0.2mol/L of ammonium acetate = 45; and (3) taking 50mL of a mixed solution of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2mol/L ammonium acetate solution, adding 0.3mL of NaOH with the mass percentage concentration of 50%, and uniformly mixing.
2.2 Method) of
Respectively and precisely measuring 0.1mL of each of the aluminum element series reference substance solution, the blank reference solution, the sample solution to be tested obtained under different extracting agents and the standard sample solution of different extracting agents, respectively and precisely adding 0.9mL of the derivative reagent, uniformly mixing, respectively injecting into a liquid chromatograph, and recording the chromatogram.
The liquid chromatography conditions were: adopting a phenyl chromatographic column with phenethyl bonded silica gel as a matrix, wherein a mobile phase is a mixture of an 8-hydroxyquinoline acetonitrile solution with the mass percentage concentration of 0.3% and an ammonium acetate solution with the concentration of 0.2mol/L according to the volume ratio of 45; the column temperature is 30 ℃; the flow rate is 1.0mL/min; the sample injection volume is 100 mu L; the excitation wavelength of the fluorescence detector was 380nm and the emission wavelength was 520nm.
And performing linear regression on the corresponding peak areas of the concentrations of the aluminum element series reference substance solutions to obtain a linear regression equation, and respectively calculating the content of the aluminum element in the solutions according to the peak areas of the sample solutions of the samples to be tested obtained at different temperatures and the standard samples at different temperatures. The recovery was calculated and the results are given in table 4.
Table 4 extractant screening:
as can be seen from Table 4, the graphite digestion instrument heating mode is preferred in the invention, and n-heptane is used as an extracting agent. The extraction is carried out by using 1ml, 2ml and 3ml of n-heptane respectively, when 1ml of n-heptane is used for extraction, a small amount of oil phase impurities which are not dissolved in the two phases are still remained between the water phase and the oil phase, and when 2ml and 3ml of n-heptane are used for extraction, the water phase and the oil phase are completely separated. The preferred n-heptane loading for this invention is 2-3mL, more preferably 3mL, with a typical chromatogram shown in FIG. 11.
In summary, the preferred preparation of the sample solution of the test sample in the present invention is: taking a polytetrafluoroethylene digestion tube, adding 9mL of a sample to be tested and 1mL of hydrochloric acid according to a volume ratio of 9 to 1, plugging, placing the tube in a graphite digestion instrument, heating the tube at 80 ℃ for 30min, heating the tube to 100 ℃ for 1h, destroying the oil-in-water structure of the fat emulsion, concentrating the solution at 120 ℃ in an oil-water separation turbid state, cooling the solution, transferring the solution by using purified water, and fixing the volume to 10mL. Adding 3mL of n-heptane, shaking uniformly, standing for layering, removing an oil phase, and filtering a water phase to obtain a sample solution of the test sample.
(III) selection of mobile phase and concentration of derivatizing reagent
1. Selection of 8-hydroxyquinoline concentration
8-hydroxyquinoline is combined with aluminum ions to form a fluorescent complex, and the content of the complex is measured by using a fluorescence detector, thereby quantifying the aluminum element. 8-hydroxyquinoline is a colored substance, and in the determination system, the 8-hydroxyquinoline exists in the dual identities of a derivative reagent and a mobile phase component, so that the influence of background is eliminated. The invention screens the concentration of 8-hydroxyquinoline acetonitrile solution: respectively selecting 0.1%,0.3%,1% and 3% of 8-hydroxyquinoline.
According to the invention, through experimental investigation, the concentration of 8-hydroxyquinoline in the composition of the mobile phase and the derivatization reagent is 0.3%, and the solvent is acetonitrile, which is combined with factors such as the response value of the sample, the durability of the method, the stability of the mobile phase and the like.
2. The peak shape of the fluorescent complex formed by combining the 8-hydroxyquinoline with the aluminum ions in the concentration of the sodium hydroxide in the derivatization reagent is better under the solution environment with the pH value of more than 7, so that the pH environment of a sample is changed by adding the sodium hydroxide solution with a certain concentration in the mobile phase, and the peak shape of the chromatographic peak is improved. The invention screens the concentration of NaOH in the derivative reagent: the concentrations of NaOH were 0,0.3%, and 0.5%, respectively.
According to experimental investigation, in combination with the chromatographic peak shape and the reduction of the introduction of foreign aluminum elements, 50% NaOH solution was selected and added to the mobile phase in a small amount so that the concentration of NaOH in the mobile phase was 0.3%.
Example 2
The method for determining the content of the impurity aluminum element in the fat emulsion injection by the high performance liquid chromatography comprises the following steps:
1. preparing a reagent:
1.1 Formulating a derivatizing reagent:
0.3% by volume of a solution of 8-hydroxyquinoline in acetonitrile: 0.2mol/L of ammonium acetate = 45; and (3) taking 50mL of a mixed solution of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2mol/L ammonium acetate solution, adding 0.3mL of NaOH with the mass percentage concentration of 50%, and uniformly mixing.
1.2 Preparing aluminum element series reference solution:
preparing standard stock solution of aluminum element: preparing standard stock solution of aluminum element with the concentration of 500 mug/L.
Preparing aluminum element series reference substance solution: an appropriate amount of standard stock solution of aluminum element is precisely measured, purified water is added for quantitative dilution, and aluminum element series reference substance solutions with the concentrations of 200 mug/L, 100 mug/L, 50 mug/L, 25 mug/L, 10 mug/L and 5 mug/L are respectively prepared.
1.3 Blank control solution):
purified water was used as a blank solution.
1.4 Mobile phase preparation:
0.3% 8-hydroxyquinoline acetonitrile solution: 0.2mol/L ammonium acetate solution = 45; taking 0.3 percent of 8-hydroxyquinoline acetonitrile solution and 0.2mol/L of ammonium acetate solution, and uniformly mixing.
2. Preparing a sample solution of a test sample:
taking a polytetrafluoroethylene material digestion tube, precisely adding 9mL of a sample to be tested and 1mL of hydrochloric acid, adding a plug, placing the tube in a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring by using purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, shaking uniformly, standing for layering, removing an oil phase, filtering a water phase, and taking a filtrate to obtain a sample solution of the sample to be tested. The following test samples are all medium-long chain fat emulsion injection (C) 6 -C 24 )。
3. Blank test article solution:
taking a polytetrafluoroethylene material digestion tube, precisely adding 9mL of purified water and 1mL of hydrochloric acid, adding a plug, placing the tube into a graphite digestion instrument, heating the tube at 80 ℃ for 30min, heating the tube to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling the product, fixing the volume to 10mL by using the purified water, adding 3mL of n-heptane for extraction, shaking the product uniformly, standing the product for layering, removing an oil phase, taking a lower-layer water phase, filtering the lower-layer water phase, and taking a filtrate to obtain a blank sample solution;
4. measurement of
The instrument comprises the following steps: shimadzu 20A high performance liquid chromatograph, shimadzu RF-20A fluorescence detector.
Respectively and precisely measuring 0.1mL of each of an aluminum element series reference substance solution, a blank reference substance solution, a sample solution to be tested and a blank sample solution to be tested, respectively and precisely adding 0.9mL of a derivative reagent, uniformly mixing, respectively injecting into a high performance liquid chromatograph, and recording a chromatogram;
the liquid chromatography conditions were: adopting a phenyl chromatographic column (CAPCELL PAK phenyl4.6mm multiplied by 250mm,5 mu m) with phenethyl bonded silica gel as a substrate, wherein a mobile phase is a mixture of 8-hydroxyquinoline acetonitrile solution with the mass percentage concentration of 0.3 percent and ammonium acetate solution with the concentration of 0.2mol/L according to the volume ratio of 45; the column temperature is 30 ℃; the flow rate is 1.0mL/min; the sample injection volume is 100 mu L; the excitation wavelength of the fluorescence detector was 380nm and the emission wavelength was 520nm.
5. Standard curve drawing and calculation
And (3) performing linear regression by subtracting the peak area of the blank reference solution from the corresponding peak area of the concentration of the aluminum element series reference solution to obtain a linear regression equation, and calculating the content of the aluminum element in the test solution according to the peak area of the test solution by subtracting the peak area of the blank test solution.
(II) precision test
Taking the aluminum element control solution with the concentration of 25 mug/L in the aluminum element series control solution, and carrying out determination by the method of determination 4 in (I), and continuously feeding samples for 6 times, wherein the results are shown in Table 5.
TABLE 5 sample introduction precision test results
As can be seen from Table 5, the method of the present invention has a sample injection precision RSD of 0.62%, and the method has a good sample injection precision.
(III) repeatability test
A total of 6 test sample solutions were prepared according to the test sample solutions of 2 in (A), and then repeated experiments were carried out according to the method of (A), with the results shown in Table 6.
TABLE 6 results of the repeatability tests
As can be seen from Table 6, the RSD of the method of the present invention is 5.5%, and the method has good reproducibility.
(IV) accuracy test (sample recovery):
aluminum element control concentrated solution: precisely weighing 25mL of aluminum element standard stock solution with the concentration of 500 mug/L into a 100mL measuring flask, and adding purified water for quantitative dilution to prepare an aluminum element reference solution with the concentration of 125 mug/L.
Sample solution of test article: taking a polytetrafluoroethylene digestion tube, precisely adding 1mL of hydrochloric acid and 9mL of a sample to be tested, plugging, placing the tube into a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring by using purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, shaking uniformly, standing for layering, removing an oil phase, filtering a water phase, and taking filtrate to obtain a sample solution to be tested.
Blank test solution: taking a polytetrafluoroethylene digestion tube, precisely adding 1mL of hydrochloric acid and 9mL of purified water, plugging, placing in a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring with purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, shaking up, standing for layering, taking a lower-layer water phase, filtering, taking a filtrate, and obtaining a blank sample solution.
Adding a standard recovery rate test sample solution: respectively and precisely measuring 0.5mL, 1mL and 1.5mL of aluminum element reference substance concentrated solution with the concentration of 125 mu g/L, respectively and precisely adding 4 mL, 3.5 mL and 3mL of purified water into polytetrafluoroethylene digestion tubes, respectively and precisely adding 4.5mL of test sample into the polytetrafluoroethylene digestion tubes, uniformly mixing, precisely adding 1mL of hydrochloric acid (3 parts of each concentration), plugging, heating in a graphite digestion instrument at 80 ℃ for 30min, heating to 100 ℃ for 1h, damaging the oil-in-water structure of fat emulsion, enabling the solution to be in a turbid state of oil-water separation, and concentrating at 120 ℃. Cool down, transfer with purified water and quantify 10mL. Adding 3ml of n-heptane for extraction, shaking uniformly, standing for layering, discarding an oil phase, and filtering a water phase to obtain a sample solution with the standard recovery rate. The sample solution with blank standard addition recovery rate is prepared in the same way by using 9mL of purified water instead of the sample.
Respectively precisely measuring an aluminum element series reference substance solution, a blank reference substance solution, a sample solution of a test article, a blank test article solution, a sample solution with a standard addition recovery rate and a sample solution with a blank standard addition recovery rate by 0.1mL, precisely adding a derivative reagent by 0.9mL, uniformly mixing, and performing determination by the method of determination 4 in the step (I), wherein the results are shown in a table 7.
TABLE 7 results of recovery measurement
As can be seen from table 7, the method of the present invention has a sample recovery of 109.4% and an RSD of 1.4% (n = 9), and the method has good accuracy.
EXAMPLE 3 practical application
The method for determining the content of the impurity aluminum element in the fat emulsion injection by using the high performance liquid chromatography comprises the following steps:
1. preparing a reagent:
1.1 Formulating a derivatizing reagent:
0.3% by volume of a solution of 8-hydroxyquinoline in acetonitrile: 0.2mol/L of ammonium acetate = 45; and (3) taking 50mL of a mixed solution of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2mol/L ammonium acetate solution, adding 0.3mL of NaOH with the mass percentage concentration of 50%, and uniformly mixing.
1.2 Preparing aluminum element series reference solution:
preparing standard stock solution of aluminum element: taking a standard solution of metallic aluminum single element (national analysis and test center for nonferrous metals and electronic materials, the concentration is 1000 mug/ml), diluting with purified water to prepare a standard stock solution of aluminum element with the concentration of 500 mug/L.
Preparing an aluminum element series reference substance solution: an appropriate amount of standard stock solution of aluminum element is precisely measured, purified water is added for quantitative dilution, and aluminum element series reference substance solutions with the concentrations of 200 mug/L, 100 mug/L, 50 mug/L, 25 mug/L, 10 mug/L and 5 mug/L are respectively prepared.
1.3 Blank control solution): purified water was used as a blank solution.
1.4 Mobile phase preparation: 0.3% by volume of a solution of 8-hydroxyquinoline in acetonitrile: 0.2mol/L of ammonium acetate = 45; taking 0.3 percent of 8-hydroxyquinoline acetonitrile solution and 0.2mol/L of ammonium acetate solution, and uniformly mixing.
2. Preparing a sample solution of a test sample:
taking a polytetrafluoroethylene material digestion tube, precisely adding 9mL of a sample to be tested and 1mL of hydrochloric acid, adding a plug, placing the tube in a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring by using purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, shaking uniformly, standing for layering, removing an oil phase, filtering a water phase, and taking a filtrate to obtain a sample solution of the sample to be tested.
As shown in Table 8, different varieties of fat emulsion injection with different batches were used as samples.
3. Blank test solution:
taking a polytetrafluoroethylene digestion tube, precisely adding 9mL of purified water and 1mL of hydrochloric acid, plugging, placing in a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring with purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, shaking up, standing for layering, taking a lower-layer water phase, filtering, taking a filtrate, and obtaining a blank sample solution;
4. measurement of
The instrument comprises the following steps: japanese Shimadzu model 20A high performance liquid chromatograph.
Respectively and precisely measuring 0.1mL of each of an aluminum element series reference solution, a blank reference solution, a sample solution to be tested and a blank sample solution to be tested, respectively and precisely adding 0.9mL of a derivatization reagent, uniformly mixing, respectively injecting into a high performance liquid chromatograph, and recording a chromatogram;
the liquid chromatography conditions were: adopting a phenyl chromatographic column (CAPCELL PAK phenyl4.6mm multiplied by 250mm,5 mu m) with phenethyl bonded silica gel as a substrate, wherein a mobile phase is a mixture of 8-hydroxyquinoline acetonitrile solution with the mass percentage concentration of 0.3 percent and ammonium acetate solution with the concentration of 0.2mol/L according to the volume ratio of 45; the column temperature is 30 ℃; the flow rate is 1.0mL/min; the sample injection volume is 100 mu L; the excitation wavelength of the fluorescence detector was 380nm and the emission wavelength was 520nm.
5. As a result, the
As shown in fig. 12, the concentration of the aluminum element series reference solution is subtracted from the corresponding peak area of the blank reference peak area to obtain a standard curve, and a linear regression is performed to obtain a linear regression equation: y =1480.66x+14529.46 2 =0.9994. Within the range of 5-200 ug/L, the linear relation is good.
Deducting the blank sample peak area from the sample solution peak area by standard curve method to obtain the content of aluminum element (C) in the sample solution 0 ) Then, the content of aluminum element in the sample to be tested is obtained according to the formula (1), and the result is shown in table 8.
Wherein, C: the content of aluminum element in the sample to be tested is g/mL
C 0 : the content of aluminum element in the sample solution of the test sample is mu g/L
C Oil : oil phase concentration of sample to be tested, g/mL
ρ Oil : oil phase density, g/mL, of test sample
Note: the oil phase concentration of the sample to be tested and the oil phase density of the sample to be tested are provided by pharmaceutical manufacturing enterprises.
TABLE 8 sample determination
As can be seen from Table 8, the method of the present application has the advantages of accurate measurement result, simple instrument and equipment, low price and easy popularization and use in laboratories.
Claims (9)
1. The method for determining the content of the impurity aluminum element in the fat emulsion injection by using the high performance liquid chromatography is characterized by comprising the following steps:
1) Preparing a reagent: preparing a derivatization reagent, an aluminum element series reference substance solution, a blank reference solution and a mobile phase respectively,
preparing the materials when in use;
2) Preparing a sample solution of a test sample: taking a polytetrafluoroethylene digestion tube, precisely adding 9mL of a sample to be tested and 1mL of an acid reagent, plugging, placing the tube into a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring with purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, shaking up, standing for layering, removing an oil phase, filtering a water phase, and taking a filtrate to obtain a sample solution of the sample to be tested; the sample to be tested is fat emulsion injection;
3) Preparing a blank test solution: taking a polytetrafluoroethylene material digestion tube, precisely adding 9mL of purified water and 1mL of acid reagent, plugging, placing in a graphite digestion instrument, heating at 80 ℃ for 30min, heating to 100 ℃ for 1h, concentrating the obtained product at 120 ℃, cooling, transferring with purified water, fixing the volume to 10mL, adding 3mL of n-heptane for extraction, shaking up, standing for layering, taking out the lower-layer water phase for filtration, taking the filtrate, and obtaining a blank sample solution;
4) And (3) determination: respectively and precisely measuring 0.1mL of each of an aluminum element series reference solution, a blank reference solution, a sample solution of a test sample and a blank test sample solution, respectively and precisely adding 0.9mL of a derivatization reagent, uniformly mixing, respectively injecting into a liquid chromatograph, and recording a chromatogram;
5) And (3) performing linear regression by subtracting the peak area of the blank control solution from the corresponding peak area of the concentration of the aluminum element series control solution according to the concentration of the aluminum element series control solution to obtain a linear regression equation, and subtracting the peak area of the blank test solution from the peak area of the test sample solution to calculate the content of the aluminum element in the test sample.
2. The method for determining the content of the impurity aluminum element in the fat emulsion injection by using the high performance liquid chromatography as claimed in claim 1, wherein in the step 1), the preparation method of the derivatization reagent comprises the following steps: according to the volume ratio, the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.3 percent, and the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.2mol/L ammonium acetate solution = 45; taking 50mL of a mixed solution of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2mol/L ammonium acetate solution, adding 0.3mL of NaOH with the mass percentage concentration of 50%, and uniformly mixing.
3. The method for determining the content of the impurity aluminum element in the fat emulsion injection by using the high performance liquid chromatography as claimed in claim 1, wherein in the step 1), the concentrations of the aluminum element series reference substance solutions are 200 μ g/L, 100 μ g/L, 50 μ g/L, 25 μ g/L, 10 μ g/L and 5 μ g/L respectively.
4. The method for determining the content of the impurity aluminum element in the fat emulsion injection by using the high performance liquid chromatography as claimed in claim 1, wherein in the step 1), the preparation method of the mobile phase comprises the following steps: according to the volume ratio, the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.3 percent, and the mass percentage concentration of the 8-hydroxyquinoline acetonitrile solution is 0.2mol/L ammonium acetate solution = 45; taking 0.3 percent of 8-hydroxyquinoline acetonitrile solution and 0.2mol/L of ammonium acetate solution, and uniformly mixing.
5. The method for determining the content of the impurity aluminum element in the fat emulsion injection by using the high performance liquid chromatography as claimed in claim 1, wherein in the step 2), the acid reagent is sulfuric acid, nitric acid or hydrochloric acid.
6. The method for determining the content of the impurity aluminum element in the fat emulsion injection by using the high performance liquid chromatography as claimed in claim 5, wherein the acid reagent is hydrochloric acid.
7. The method for determining the content of the impurity aluminum element in the fat emulsion injection by using the high performance liquid chromatography method according to claim 1, wherein in the step 3), the liquid chromatography conditions are as follows: adopting a phenyl chromatographic column with phenethyl bonded silica gel as a matrix, wherein the column temperature is 30 ℃; the flow rate is 1.0mL/min; the sample injection volume is 100 mu L; the excitation wavelength of the fluorescence detector was 380nm and the emission wavelength was 520nm.
8. The method for determining the content of the impurity aluminum element in the fat emulsion injection by the high performance liquid chromatography according to any one of claims 1 to 7, wherein the fat emulsion injection is C 6 -C 24 The medium-long chain fat emulsion injection, or the fat emulsion injection containing the fat emulsion amino acid (17) and the glucose (11%), or the fat emulsion injection containing the medium-long chain fat emulsion amino acid (16) and the glucose (16%).
9. Use of the method of any one of claims 1 to 7 for determining the elemental aluminium content as an impurity in a fat emulsion injection.
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